Delta-like ligand 4 (Dll4) expressed in tumor cells plays a key role to promote tumor growth of numerous cancer types. Based on a novel anti-human Dll4 monoclonal antibody (61B), we developed a 64Cu-labeled probe for positron emission tomography (PET) imaging of tumor Dll4 expression. In this study, 61B was conjugated with the 64Cu-chelator DOTA through lysine on the antibody. Human IgG (hIgG)-DOTA, which did not bind to Dll4, was also prepared as a control. The Dll4 binding activity of the probes was evaluated through the bead-based binding assay with Dll4-alkaline phosphatase. The resulting PET probes were evaluated in U87MG glioblastoma and HT29 colorectal cancer xenografts in athymic nude mice. Our results demonstrated that the 61B-DOTA retained (77.2 ± 3.7) % Dll4 binding activity of the unmodified 61B, which is significantly higher than that of hIgG-DOTA (0.06 ± 0.03) %. Confocal microscopy analysis confirmed that 61B-Cy5.5, but not IgG-Cy5.5, predominantly located within the U87MG and HT29 cells cytoplasm. U87MG cells showed higher 61B-Cy5.5 binding as compared to HT29 cells. In U87MG xenografts, 61B-DOTA-64Cu demonstrated remarkable tumor accumulation (10.5 ± 1.7 and 10.2 ± 1.2 %ID/g at 24 and 48 hour post injection, respectively). In HT29 xenografts, tumor accumulation of 61B-DOTA-64Cu was significantly lower than that of U87MG (7.3 ± 1.3 and 6.6 ± 1.3 %ID/g at 24 and 48 hour post injection, respectively). The tumor accumulation of 61B-DOTA-64Cu was significantly higher than that of hIgG-DOTA-64Cu in both xenografts models.

 

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